Project Summary/Abstract Glycans play important roles in biological systems and many diseases, often through their specific interactions with other biomolecules such as glycan-binding proteins (GBPs). In the past decades, the systematic study of protein-glycan interactions has been greatly improved by the development of glycan microarray technology, in which a library of glycan structures is immobilized by a microarray printer onto solid surfaces such as glass slides and interrogated with fluorescently labeled GBPs. The binding specificities of GBPs can be quickly deduced from the fluorescent image acquired by a microarray scanner. Despite its success, the current glycan microarray technology is seriously limited in several aspects. First, the number of glycans included in current glycan microarrays is limited by chemical/enzymatic synthesis. Even in the most popular glycan microarray provided by the Consortium for Functional Glycomics, only a small fraction (~600 glycans) of glycome is represented. This number is growing quickly owing to novel chemoenzymatic synthesis and novel methods to utilize natural glycans; however, using current glycan microarray platforms, it would be difficult to accommodate more than 1,000 glycans. Second, while glycan microarray is considered a high throughput platform due to the large number of glycans that can be analyzed simultaneously, it actually suffers from a bottleneck in processing that requires a manual alignment of a grid in the fluorescent image to quantify the fluorescent intensity at each individual spot. Thus, processing of many samples such as patient serum samples is a very labor intensive and slow process. Third, despite the simple concept, glycan microarray technology is limited to a number of very specialized laboratories due to the high cost of instrumentation including microarray printer and scanner. To address these challenges, we propose a novel approach termed Next Generation Glycan Microarray (NGGM) enabled by Next Generation Sequencing (NGS). In the new approach, we will present glycan microarray as a mixture of glycans and/or glycoconjugates that are coded with oligonucleotide sequences. The immunoprecipitation of glycans with GBPs will select specifically bound glycans with DNA codes. The codes can be decoded using the powerful quantitative NGS technology to elucidate the relative binding specificities of glycan structures to GBPs. The sequence reads will be converted to binding specificities of GBPs. We expect that this new approach will solve the problems of capacities, throughput, easy accessibility and affordability. It will greatly lower the threshold for the general scientific community to study protein-glycan interactions.